This invention relates to a method for the enrichment of dendritic cells from the peripheral blood of a mammal, to purified dendritic cell populations, and to kits useful for enrichment of dendritic cells.
Dendritic cells are antigen-presenting cells that are found in all tissues and organs, including the blood. Specifically, dendritic cells present antigens for T lymphocytes, i.e., they process and present antigens, and stimulate responses from naive and memory T cells. In addition to their role in antigen presentation, dendritic cells directly communicate with non-lymph tissue and survey non-lymph for an injury signal (e.g., ischemia, infection, or inflammation) or tumor growth. Once signaled, dendritic cells initiate the immune response by releasing IL-1 which triggers lymphocytes and monocytes.
Dendritic cells can play either a positive or negative role in various immunologically related situations. For example, dendritic cells are thought to be involved in transplant rejection and autoimmune diseases, and it may be desirable to block their function in such situations. On the other hand, various immunodeficiencies, e.g., towards tumors, may result from deficient dendritic cells. In addition, there is evidence that HIV can infect certain populations of dendritic cells.
It is desirable to obtain substantially pure populations of dendritic cells, e.g., in order to exploit the important role of dendritic cells in various immunotherapies. Dendritic cell isolation is particularly difficult, however, because it is believed that they are very low in frequency in blood and other tissues, and because there is no reported surface marker expressed by fresh and cultured dendritic cells which distinguishes it from monocytes. Conventional methods for isolation which enrich subpopulations of cell mixtures include, e.g., density gradient separation, fluorescence activated cell sorting, immunological cell separation techniques such as panning, complement lysis, rosetting, magnetic cell separation techniques, and nylon wool separation. Different patterns of expression of cell surface antigens have been used in some cases to identify different cell types. Certain disadvantages of many of these reported methods are that they can be time-consuming, labor-intensive, costly, require large amounts of reagent, can result in low specificity, low sensitivity, contaminated mixtures, poor and/or inaccurate separation, loss of desired cells, or can change the properties, functions, or viability of the desired cells. Thus, prior methods generally are inefficient, time-consuming, expensive and do not optimize for pure populations.
It is an object of the invention to provide an easy, effective, and inexpensive method for isolating dendritic cells from tissue, e.g., peripheral blood, of a mammal.
It is yet another object of the invention to provide a method for isolating dendritic cells which are viable and substantially unaltered in cell function.
It is yet another object of the invention to provide a method for isolating dendritic cells which are substantially pure.
It is yet another object of the invention to provide a method for isolating dendritic cells which can be used in various immunotherapeutic regimens.
It is yet another object of the invention to provide a method for isolating dendritic cells which express high levels of CD14.
It is yet another object of the invention to provide a method for isolating dendritic cells from monocytes.
It is yet another object of the invention to provide a method for isolating dendritic cells from tissue, e.g., peripheral blood, of a mammal which are unactivated.
Still another object of the invention is to provide a method for isolating monocytes from the tissue, e.g., peripheral blood, of a mammal which are unactivated.
According to the invention, a method for the enrichment of dendritic cells from the peripheral blood of a mammal is provided. Peripheral blood having mononuclear cells from a mammal is provided, and the mononuclear cells are separated from the peripheral blood. These mononuclear cells are separated into a first cell population having substantially lymphocytes, e.g., T cells, NK cells, B cells or mixtures thereof, and a second cell population having substantially myeloid cells. These myeloid cells are separated into a third cell population having substantially monocytes and a fourth cell population having substantially dendritic cells.
In certain embodiments, the separation of the mononuclear cells into a first cell population having substantially lymphocytes and a second cell population having substantially myeloid cells comprises contacting the mononuclear cells with antibodies against the lymphocytes so as to form an antibody-lymphocyte complex, and selectively separating the antibody-lymphocyte complex from the myeloid cells. The antibodies used, e.g., monoclonal antibodies, are directed against one or more antigens which are expressed by one or more of the lymphocytes. For example, T cell antibodies include anti-CD3 antibodies, anti-CD8 antibodies, and mixtures thereof; NK cell antibodies include, e.g., anti-CD16/56 antibodies; and B cell antibodies include, e.g., anti-CD19 or anti-CD20 antibodies.
In certain embodiments, the antibody-lymphocyte complex that is formed is selectively separated from the myeloid cells by contacting the antibody-lymphocyte complex and the myeloid cells with a matrix such that the antibody-lymphocyte complex is substantially retained by the matrix, e.g., greater than 20%, 40%, 60%, 80%, 90%, 95%, 98%, or 99% retained, and the myeloid cells are substantially not retained by the matrix, e.g., greater than 20%, 40%, 60%, 80%, 90%, 95%, 98%, or 99% not retained.
Preferably, the antibody-lymphocyte complex further comprises magnetic beads, e.g., superparamagnetic microparticles. The magnetic beads can be attached, e.g., to the antibody or to the lymphocyte or to both. In embodiments in which the antibody-lymphocyte complex has magnetic beads, separation of such a complex from the myeloid cells preferably comprises contacting the myeloid cells and the complex with a magnetic matrix, e.g., magnetized steel wool, such that the antibody-lymphocyte complex having the magnetic beads is substantially retained by the magnetic matrix and the myeloid cells are substantially not retained by the magnetic matrix.
A variation of this method is separating the mononuclear cells into a first cell population having substantially lymphocytes and a second cell population having substantially myeloid cells by centrifugation, e.g., density gradient centrifugation.
In certain embodiments, the separation of the mononuclear cells into a third cell population having substantially monocytes and a fourth cell population having substantially dendritic cells comprises contacting the myeloid cells with antibodies against the dendritic cells so as to form an antibody-dendritic cell complex, and selectively separating the antibody-dendritic cell complex from the monocytes. The antibodies used, e.g., monoclonal antibodies, are directed against one or more antigens which are expressed by the dendritic cells, e.g., anti-CD2 antibodies, anti-CD5 antibodies or mixtures thereof. In certain embodiments, the myeloid cells are cultured prior to contacting the myeloid cells with the antibodies, and anti-CD83 antibodies are used.
In certain embodiments, the antibody-dendritic cell complex that is formed is selectively separated from the monocytes by contacting the antibody-dendritic cell complex and the monocytes with a matrix such that the antibody-dendritic cell complex is substantially retained by the matrix and the monocytes are substantially not retained by the matrix. Preferably, the retained antibody-dendritic cell complex is then eluted from the matrix.
Preferably, the antibody-dendritic cell complex further comprises magnetic beads. In such embodiments, separation of the antibody-dendritic complex from the monocytes preferably comprises contacting the monocytes and the antibody-dendritic cell complex having the magnetic beads with a magnetic matrix such that the antibody-dendritic cell complex having the magnetic beads is substantially retained by the magnetic matrix and the monocytes are substantially not retained by the magnetic matrix. Preferably, the retained antibody-dendritic cell complex is then eluted from the matrix, e.g., by demagnetizing the matrix, e.g., by removing the matrix from the magnetic field.
Preferably, the dendritic cells in the fourth cell population are greater than about 60%, 70%, 80%, 90%, 95% 98%, or 99% pure. Preferably, the monocytes in the third cell population are greater than about 70%, 80%, 90%, 95%, 98%, or 99% pure. Preferably, the monocytes in the third cell population and/or the dendritic cells in the fourth cell population are substantially unactivated.
Other aspects of the invention are enriching for dendritic cells from the peripheral blood mononuclear cells or from the myeloid cells of a mammal.
Another aspect of the invention is a method for the enrichment of dendritic cells from the peripheral blood of a mammal comprising selecting cells from the peripheral blood which do not express antigens CD3, CD16/56 and CD19 or CD20, and which do express antigen CD2, CD5, CD83, or mixtures thereof, and which preferably, also express antigen CD14.
The invention also includes a method for the enrichment of dendritic cells from tissue of a mammal. Tissue having mononuclear cells from a mammal is provided. The mononuclear cells are separated from the tissue. The mononuclear cells are separated into a first cell population having substantially lymphocytes and a second cell population having substantially myeloid cells. The myeloid cells are separated into a third cell population having substantially monocytes and a fourth cell population having substantially dendritic cells.
Another aspect of the invention is the products obtained from the above described methods.
Another aspect of the invention is a substantially purified population of mammalian dendritic cells, e.g., greater than about 80%, 90%, 95%, 98%, or 99% pure, in which the dendritic cells express antigen CD14. In certain embodiments, the dendritic cells also express antigen CD2 and/or CD5 and/or CD83.
Another aspect of the invention is a kit useful for enriching for dendritic cells from blood. The kit includes, e.g., anti-CD2 antibodies, anti-CD5 antibodies, anti-CD83 antibodies, or mixtures thereof, and lymphocyte antibodies. The lymphocytes include, e.g., T cells, NK cells, B cells and mixtures thereof. Preferably, the T cell antibodies include anti-CD3 antibodies, anti-CD8 antibodies, or mixtures thereof; the NK cell antibodies include, e.g., anti-CD16/56 antibodies; and the B cell antibodies include, e.g., anti-CD19 or anti-CD20 antibodies.
Variations include, e.g., the kit also having magnetic beads, e.g., superparamagnetic microparticles, which are either complexed to the antibodies or are separate. Other variations include the kit having matrix material, preferably provided in a column, and preferably capable of being magnetized by exposure to a magnetic field, e.g., the matrix can be steel wool.
Another aspect of the invention is a vaccine composition for treating cancer in a mammal comprising a therapeutically effective amount of dendritic cells wherein the dendritic cells express antigen CD14. In certain embodiments, the dendritic cells are treated with cancer-specific antigen so as to stimulate host immunity to the cancer when the vaccine composition is administered to a mammal.
Yet another aspect of the invention is a method for treating a mammal for cancer. A mammal in need of treatment for cancer is provided. A vaccine composition comprising a therapeutically effective amount of dendritic cells wherein the dendritic cells express antigen CD14, is provided. The vaccine composition is administered to the mammal such that treatment of the cancer occurs.
Earlier methods for the preparation of dendritic cells have depended on the beliefs that monocytes are CD14+, CD11b+, CD18+, whereas dendritic cells are CD14xe2x88x92, CD11bxe2x88x92, CD18xe2x88x92, or in the case of CD14, dim and much lower than monocytes, and that both cell populations are CD2xe2x88x92 and CD5xe2x88x92. These earlier methods generally yield purified preparations of dendritic cells that represent about 0.1-0.3% of peripheral blood mononuclear cells. This invention, however, indicates that freshly isolated dendritic cells express high levels of CD2, CD5, CD14, CD11b and CD18, whereas freshly isolated monocytes express only high levels of CD14, CD11b and CD18, and do not express either CD2 or CD5. Furthermore, CD14, CD11b and CD18 are downregulated or lost on incubation of dendritic cells, particularly in the universally used fetal calf serum as medium additive.
This invention is partially based on the fact that certain low density mononuclear cells are largely CD33+ monocytes and dendritic cells. As with prior methods, contaminating T cells, B cells and NK cells are removed with anti-CD3, anti-CD20 and anti-CD16 antibodies. In the new method of this current invention, anti-CD2 (or anti-CD5) beads are used to positively select dendritic cells. In addition, the culturing is performed in pooled human serum rather than fetal calf serum to avoid activation. The purified dendritic cells exhibit greater mixed lymphocyte reactivity and express higher amounts of class II MHC antigens than purified monocytes, show typical dendritic cell morphology on electron microscopy, and react with X-11 and anti-CD83, antibodies that react with dendritic cells but not monocytes.
The advantages of the invention include, e.g., (i) greater yield (up to about 30-fold greater) compared with prior methods; (ii) greater purity of the dendritic cells ( greater than 95% compared with about 50-60% using prior methods); (iii) more representative population of dendritic cells than obtained from prior methods; (iv) the ability to isolate non-activated dendritic cells capable of all functions, which is not possible with previous methods; (v) much shorter time for isolation (about 75 minutes vs. about 5-36 hours with prior methods); and (vi) applicable to skin, lymph nodes and other tissues.
The above and other objects, features and advantages of the present invention will be better understood from the following specification when read in conjunction with the accompanying drawings.